Iron (Fe) is an essential microelement for plants. We have recently shown that ascorbate plays a central role in Fe transport to seeds by mediating Fe3+ reduction for the uptake of Fe2+. Moreover, the ascorbate efflux activity at the embryo surface was crucial in this process. We have used a yeast complementation strategy to isolate ascorbate efflux transporters, by expressing an Arabidopsis cDNA library in the Δfre1 mutant that lacks ferric reductase activity and is unable to grow in Fe limiting conditions. The expression of two cDNAs named MATE and GAL is able to rescue the growth defect of Δfre1by mediating efflux of ascorbate in the medium, reconstituting a ferric reduction activity. Therefore, we have studied the roles of putative ascorbate efflux transporters, MATE and GAL, in Fe transport and homeostasis in Arabidopsis thaliana. In this study, we have identified the MATE protein as a vacuolar transporter potentially involved in loading ascorbate to the vacuole to reduce intra-vacuolar Fe. This transport activity appears to be crucial to remobilize Fe during germination and to participate in the response to Fe deficiency in the rest of the plant, as revealed by the phenotypical analyses of knock out mutant plants. The GAL protein is localized to the plasma membrane where it could potentially catalyze the efflux of ascorbate toward the apoplast to remobilize apoplastic Fe pools. Indeed, GAL knock out mutants are highly sensitive to Fe deficiency and disturbed in the sensing of Fe nutritional status in Fe-replete conditions. In conclusion, the two putative ascorbate transporters identified in this study appear to be involved inthe iron homeostasis by regulating the movement of subcellular Fe pools. This research has contributed to discover and highlight the link between iron and ascorbate metabolism and transport.